Project Summary/Abstract Environmental exposures to industrial chemicals, such as carbon tetrachloride (CCl4) causes toxicant-induced steatohepatitis (TASH) characterized by fatty liver, inflammation, and fibrosis. Although susceptibility to occupational and environmental TASH may be modified by genetic predisposition (SNPs) and nutritional factors, our central hypothesis are that obesity and non-alcoholic fatty liver (NAFL) increase the severity of TASH by (1) Inducing the activity of cytochrome P450 2E1, (2) Increasing the generation of reactive oxygen species (ROS) by activating NADPH oxidases (NOXs), and (3) Inducing dysbiosis of the gut microbiome and increased intestinal permeability. The goal is to determine the mechanism by which obesity and CCl4 synergistically facilitate progression of TASH to liver cirrhosis, and develop new approaches to identifying toxicants that induce TASH. The following specific aims have been developed: (AIM1) To study the effect of ?fast food diet? (FFD)- induced obesity and NAFL on CCl4-induced TASH. We propose to use improved diet-induced and genetic mouse models, including mice expressing human CYP2E1 gene and knock-in PNPLA3 mice (expressing the human SNP associated with NAFL). Since hCYP2E1 mice are more susceptible to toxicants, we anticipate that production of Nox1 and Nox4, release of pro-inflammatory cytokines by activated Kupffer cells, and development of liver fibrosis will closely recapitulate the pathology observed in patients with NASH/TASH. The specific role of NOX 1 and 4 in this TASH model will be assessed by pharmacological inhibition. (AIM2) To investigate the role of dysbiosis in transgenic mice subjected to fecal microbiota transplantation (FMT) from patients with NASH or matched normal controls from a well phenotyped cohort. We propose that the microbiome from NASH patients will render mice more sensitive to CCl4, while the transfer of ?healthy? microbiota will decrease liver fibrosis in these mice. We will then do a therapeutic intervention by FMT of normal flora into a mouse with liver fibrosis to assess reversal of TASH. (AIM3) To translate our findings in mice to humans, we will utilize a ?human liver in a dish? (exVive3D? Livers, Organovo), a 3D culture composed of 4 primary hepatic cell types that maintains architectural and functional features of the human liver for greater than 40 days. These cultures will be subjected to Superfund toxicants and then assessed for TASH. The effects of potential drugs, such as Nox inhibitors, will be assessed in these 3D culture models of TASH. Overall, we will develop a high through-put system for ex vivo drug screening by measuring hepatotoxicity of Superfund toxicants and effectiveness of therapeutic interventions.